Changes in serum uric acid concentrations after caesarean section using methoxyflurane. In a study comparing the changes in serum uric acid concentration after methoxyflurane anaesthesia in 10 patients subjected to either Caesarean section or minor general surgical procedures, a significantly greater increase in serum uric acid concentration occurred in those patients undergoing Caesarean section. These results were compared with results from a control group of five patients undergoing Caesarean section, anaesthetized without methoxyflurance and with a second control group of five patients having uncomplicated spontaneous deliveries. Reasons are presented to suggest that although obstetric patients are unlikely to be more vulnerable to methoxyflurane nephrotoxicity, those with pre-eclamptic toxaemia may be more at risk.


The speed of onset and potency of althesin. The speed of onset of anaesthesia with Althesin has been compared with that of other intravenous anaesthetics. Like thiopentone, Althesin appears to be a truly rapidly acting drug, producing sleep in one arm-brain circulation time. The relative potencies of the drugs studied were approximately Althesin 60 mulitre/kg equivalent to thiopentone 4 mg/kg, equivalent to methohexitone 1.2 mg/kg, although by some criteria Althesin 80 mulitre/kg is required to produce an effect equivalent to thiopentone 4 mg/kg.


Prolonged block with recovery after extradural analgesia for labour. A patient had a prolonged block after continuous extradural analgesia in labour. The block was unilateral, but 60 min after a second block there was bilateral sensory and motor loss. The original unblocked side did not recover fully until more than 60 hr. The local anaesthetic agent was 18 ml of bupivacine 0.5% plain.


A study of the changes in serum potassium concentration with suxamethonium using different anaesthetic agents. Serum potassium concentrations were measured after administration of suxamethonium (1 mg/kg body wt.) in 101 patients in whom anaesthesia was induced by one of five different techniques. There was a maximum increase in serum potassium of 21.4% following induction with trichloroethylene compared with 4.4% with nitrous oxide/oxygen. There was only small increases in serum potassium with halothane and chloroform. In an additional 10 patients who received tubocurarine (3 mg) before induction of anaesthesia with thiopentone, the maximum increase in serum potassium was 10.6% following suxamethonium. It is concluded that the increase in serum potassium following induction of anaesthesia is the result of a combined effect of the anaesthetic agent and suxamethonium.


Anaphylaxis to suxamethonium. Two case reports. Anaphylaxis to suxamethonium in patients who gave no history of allergy is presented in this report. The diagnosis of anaphylaxis was based on a rapid onset of tachycardia, arterial hypotension, erythematous patches and oedema, and a positive reaction to a sensitivity test.


The pain clinic. An interdisciplinary team approach to the problem of pain. The development of interdisciplinary pain clinics is discussed; the organization and staffing is described, and an outline plan is presented. It is emphasized that interdisciplinary pain clincis are the best means of medical care for patients suffering from chronic pain.


Comparative site of action of various anaestetic agents at the mammalian myoneural junction. Pre- and postjunctional effects of three ethers (enflurane, diethyle ether, and methoxyflurane) and three non-ethers (chloroform, halothane, and trichloroethylene) were studied in the rat phrenic nerve diaphragm preparation using standard microelectrode recording techniques. Depression of postjunctional function included depression of the amplitude of miniature endplate potentials, inhibition of suxamethonium induced depolarization of the muscle endplate, prolongation of duration of the endplate potential, and increase in threshold for generation of the muscle action potential. The last two effects were more marked for the ethers than for the non-ethers. Effects on pre-junctional function included a slight increase in fluctuation of the endplate potential (EPP) amplitude associated with the thers and chloroform, a faster rate of decline of EPP amplitude during a tetanus in presence of the ethers, and prolongation of the normal facilitatory period during paired stimulation in the presence of chloroform. Chloroform had no effect on the rate of decline of EPP amplitude during tetanic stimulation and the ethers had no effect on the facilitatory period during paired stimulation. These results indicate that volatile anaesthetic agents depress synaptic transmission by acting on multiple sites, and that the pattern of this depression is different for each drug or group of similar drugs.


The use of ultrasonic energy to vaporize anaesthetic liquids. Ultrasonic energy, provided by a commercially available water vaporizer, was used to vaporize methoxyflurane, halothane, and chloroform. The vaporizer was placed directly in the respiratory line, and the anaesthetic liquid was vaporized one drop at a time. Anaesthesia was maintained for periods up to 6 hr in horses, ponies, calves, sheep, dogs, and one pig. This method of vaporizing liquids is applicable to a wide variety of anaesthetics. It is easily controlled, and the same vaporizer can be used with a wide range of sizes of subjects. The vaporizer can be placed directly in the respiratory circuit, and adds almost no resistance to breathing.


The acute effect of pulmonary burns on lung mechanics and gas exchange in the rabbit. To determine the role of the thermal element in the production of lung injury uncomplicated by the inhalation of fumes and soot, 18 anaesthetized, paralysed and ventilated rabbits were subjected to a steam burn of the respiratory tract. Transpulmonary pressure (difference between airway and pleural pressure) was recorded under static conditions between functional residual capacity and 40 ml above this point and the values obtained were used to calculate lung compliance. Static compliance decreased progressively with time in each of the experimental animals, the maximum decrease occurring within the 1st hour. Ten of the 18 animals died within 4 hr of the burn. There was little impairment of gas exchange. This data would suggest the clinical value of the early measurement of lung compliance in the evaluation of thermal pulmonary burns. Postmortem studies showed the presence of pulmonary oedema with intra-alveolar peribronchial and perivascular haemorrhages. The lesions were most prominent centrally, apparently because the dissipation of the heat resulted in less damage to the peripheral areas of the lung. Pulmonary megakaryocytosis was noted in seven of the experimental animals. This may have been a non-specific reaction to lung trauma.


Studies with the "Severinghaus" PCO2 electrode I: electrode stability, memory and S plots. A study of the Radiometer "Severinghaus" Pco2 electrode has been made in order to investigate its stability and reproducibility when exposed to a series of inknown gas samples of various carbon dioxide concentrations. Electrodes were found to exhibit a pronounced memory effect, which depended upon the Pco2 of the last sample to which they had been exposed if the exposure had been for more than a few minutes. When a number of electrodes were exposed to a single reference gas at all times, except for the few minutes required to measure the Pco2 of an unknown sample, the electrical output of the electrode was unexpectedly stable and reproducible. This implies a high degree of measurement accuracy. If the reference gas to which the electrode is usually exposed is of low carbon dioxide concentration, the electrode displays its highest degree of stability and reproducibiltiy. The use of low carbon dioxide content reference gases is not always convenient, however, because they increase the response time of the electrode. Reference gases of high carbon dioxide content induce unstable electrode behaviour, and so it is recommended that gases higher than 5% v/v Co2 are not chosen for this purpose.


Studies with the "Severinghaus" Pco2 electrode II: CO2 measurement using a single control analyser. A simple, single calibration point, single control Pco2 analyser is described. Results are presented to show that such an analyser will provide extremely accurate and reproducible results over a limited range around the calibration point, and this technique has been applied to the measurement of the apparent blood-gas and water-gas difference for a Pco2 electrode. The accuracy and working range of this simple system has been compared with a two-calibration-point, two-control analyser similar to commercial systems and results are presented to show that the more complicated system, although accurate over a wider range of Pco2, did not produce accurate results when the Pco2 of test samples lay outside the calibration range. Reasons are presented to explain this unexpected behaviour.


The effect of tubocurarine on indirectly elicited train-of-four muscle response and respiratory measurements in humans. The effect of tubocurarine on the respiratory function of conscious, non-medicated volunteers was correlated with the degree of neuromuscular blockade measured with the train-of-four technique (the evoked response of the adductor pollicis brevis muscle to trains of four supramaximal ulnar nerve stimuli at 2 Hz, repeated once every 10 sec). Respiratory frequency, tidal volume, vital capacity, inspiratory force and peak expiratory flow rate were measured, (a) before administration of tubocurarine, (b) when the ratio of the amplitude of the fourth response to the amplitude of the first response of the train-of-four reached 60%, and (c) during recovery of the ratio until the control (100%) value was reached. Respiratory frequency, tidal volume and peak expiratory flow rate were not altered. Vital capacity and inspiratory force were both reduced significantly at the 60% level and the former also at the 70% level when compared with the control. The magnitude of change in all variables is of minor clinical importance, however, since the lowest measured values are well above acceptable minimum limits required for adequate respiratory function.


An investigation in man into the relative potency of lignocaine, bupivacaine and etidocaine. The relative potency of lignocaine, bupivacaine and etidocaine was estimated by forearm skin weals in four volunteers. Potency was estimated as the time to half-recovery of a sharp sensation in response to a needle-prick. The increase in duration of action with increased concentration was greater for bupivacaine and etidocaine as compared with lignocaine. At the same concentrations, bupivacaine had a longer duration of action than etidocaine. However, etidocaine 1% had a longer duration of action than bupivacaine 0.5%.


An electromyographic study of muscle relaxants in man. Supramaximal paired stimuli were applied to the ulnar nerve, and the amplitude of the muscle action potential evoked in the abductor digiti minimi by the second member of the stimulus pair (test response) was compared with that evoked by the first component (conditioning response). The interval between the two components of the stimulus pair (the pair interval) was increased stepwise from 7 to 100 msec and a curve (recovery curve) was obtained by relating the changes in pair interval to the difference in amplitude of the test and conditioning responses. Alterations of the recovery curve (RC) during partial paralysis by muscle relaxants were investigated in healthy adult patients under the lightest plane of general anaesthesia. The control curve obtained in 32 subjects before the administration of a muscle relaxant drug was characterized by slight depressions at very short intervals of paired stimuli, followed by a slight potentiation at 20-100 msec. With non-depolarizing relaxants, RC altered to the characteristic pattern of potentiation at very short intervals of stimuli, followed by a notable depression at longer intervals. In depolarizing blocks with small doses of suxamethonium, the depression of RC at short intervals in the control was enhanced and the pattern of RC was different from that of non-depolarizing agents. When desensitization blocks were instigated by the i.v. administration of suxamethonium, the RC patterns were similar to those of competitive agents.


Tympanic temperature during labour and parturition. Tympanic temperature was monitored during labour and delivery in six primigravidae and five multigravidae. Temperature increased temporarily with each contraction and progressively during the course of labour. The increases, which were greater in primiparae than in multiparae (mean cumulative increase 1.46 degrees C and 0.51 degress C respectively), reflect the metabolic expenditures associated with contraction of uterine and skeletal muscles.


Use of entonox plus carbon dioxide in the dental surgery. The introduction of Entonox has made it possible to guarantee full oxygenation of the inspired gas of outpatients having nitrous oxide anaesthesia. The addition of 1-2% halothane enables good conditions to be obtained for minor surgery such as dental extraction. The use of an Entonox mixture containing 7% carbon dioxide to stimulate ventilation in patients receiving anaesthesia with nitrous oxide, oxygen and halothane is described.


Reaction to ketamine: anaphylactoid or anaphylactic? A 3-year-old child developed extensive macular rash after ketamine. The mechanism of this reaction was investigated by the Prausnitz-Kutzner (P-K) test. It demonstrated that the histamine release was not the result of an anaphylactic reaction, but rather a direct pharmacological effect of the drug. The result suggests that this child can receive ketamine in the future without any severe manifestations. The mechanisms of histamine release are discussed and the treatment of its clinical manifestations is reviewed.


An easily missed foreign body in the respiratory passages. A case is described of a 22-year-old man who had inhaled a piece of shattered windscreen glass following a road traffic accident. The indications for bronchoscopy are discussed and the literature reviewed.


Lung function in childhood. I. The forced expiratory volumes in healthy children  using a spirometer and reverse plethysmograph. Measurements of FEV0-5, FEV0-75 and FEV1 and FVG have been made on 241 healthy schoolchildren using a spirometer and a reverse plethysmorgraph system. The results obtained by the two systems were very similar. The FEV0-75 was selected as the most appropriate forced expiratory volume for routine clinical use. Normal data charts for FEV0-75 and FVG are presented.


The diagnosis of asbestosis.

The diagnosis of asbestosis. Lung biopsy in 16 patients with abnormal lung function and who had been exposed to asbestos revealed asbestosis in 4 with considerable radiological evidence of the disease, in of 4 with minimal radiological changes and in none of 8 with completely clear radiographs. Thus biopsy for the diagnosis of this condition should be avoided if the chest radiograph shows no evidence of it.


Leiomyosarcoma of the mediastinum.

Leiomyosarcoma of the mediastinum. A case of leiomyosarcoma of the posterior mediastinum is presented; 2 other malignant and 7 benign smooth muscle tumours of the mediastinum have been collected from the literautre. The tendency for blood-borne and local recurrence in the leiomyosarcomas is demonstrated. Various theories for the origin of these tumours are examined.


Lung function in childhood. 2. Thoracic gas volumes and helium functional residual capacity measurements in healthy children. The helium dilution functional residual capacity (FRC) and the total gas volume by plethysmograph (TGV) were measured on 236 healthy London schoolchildren. There was no difference between the results obtained by the boys and girls. The results of the TGV were higher than the FRC values. From the results, normal data charts have been devised for clincial use.


Skin tests and clinical features of asthma. A report to the research committee of the British Thoracic and Tuberculosis Association. Patients with asthma aged 20-49 years recruited from chest clinics in Great Britain were classified into Groups 0, 1, 2 and 3+ according to the number of positive skin reactions to skin tests with 23 allergens. Intake was regulated in order to arrive at approximately equal numbers and a similar age and sex distribution in each of the skin test groups. The clinical data recorded on questionnaires were studied in relation to the number of positive skin tests. There were few significant differences between the four skin test groups or between patients in Group 3+ who also gave a clinical history of hypersensitivity to external allergens and those patients in Group 0 who gave no such history. No correlation was found between the age of onset of asthma and the number of parents and siblings with allergic diseases. Exercise induced asthma and predominantly nocturnal attacks were not associated with other distinctive clinical features. There was a significant association between the history of pollen sensitivity and positive skin and nasal tests with grass pollen. A significant association was also found between the clinical history of house dust allergy and the skin test, but not the nasal test, with D. pteronyssinus.


Oesophagobronchial fistual presenting in the adult. A case of congenital oesophagobronchial fistula presenting in adult life and treated successfully by surgical excision is reported. The most unusual feature is its late presentation and high origin.


Biological aspects of twin pregnancy in Dundee. A review is presented of 712 twin pregnancies which occurred in Dundee between 1956 and 1971. It shows a falling age of twin reproduction, a changing ratio of mono- to dizygosity and the poor growth rates of both types. Birth weight variations suggest that "crowding" in the uterus may affect the individual growth of each twin, but the combined weight of twins continues to increase to term and shows no obvious sign of restriction.


Blood coagulation profile in Indian patients with pre-eclampsia and eclampsia. Twelve Indian patients with pre-eclampsia, 15 with eclampsia and 15 with normal pregnancy in the third trimester were investigated. A systemic bleeding diathesis was encountered in two patients with eclampsia and in none with pre-eclampsia; two patients with pre-eclampsia, however, had excessive uterine haemorrhage. Coagulation studies showed statistically significant prolongation of thrombin time, elevation of serum fibrinogen degradation products (FDP) and hypofibrinogenaemia in patients with pre-eclampsia as well as eclampsia. In patients with eclampsia, significant thrombocytopenia also occurred. Euglobulin lysis time showed no significant change in patients with pre-eclampsia and eclampsia. There was no significant difference in the coagulation profile between patients with eclampsia and pre-eclampsia, except for more hypofibrinogenaemia in the former. The laboratory findings suggest the occurrence of intravascular coagulation in patients with pre-eclampsia and eclampsia.


Total urinary oestrogen excretion during normal menstrual cycle and normal pregnancy in Nigerians. Total oestrogens were measured during normal menstrual cycles and during pregnancy in healthy Nigerian women. During the menstrual cycle, maximum excretion was consistently observed in the luteal phase in contrast to findings in European and American populations. During pregnancy, oestrogen excretion was similar to that reported amongst Europeans only in early pregnancy; oestrogen excretion did not show the usual rapid rise in late pregnancy.


Scanning electron microscopy of normal and abnormal exfoliated cervical squamous cells. The surface ultrastructure of normal, malignant and dyskaryotic exfoliated cervical squamous cells was examined with the Scanning Electron Microscope (SEM). Normal and malignant cells were found to have very similar surfaces to those seen on similar cells in a biopsy specimen. Dyskaryotic cells, however, showed a large range of surface features and dyskaryotic cells that looked identical at light microscopy could be subdivided into three groups by examining their ultrastructural features.


Factors influencing the pregnancy and complication rates with human menopausal gonadotrophin therapy. We report the results obtained with a standard system of gonadotrophin therapy. Seventy-seven consecutive patients were given 322 treatment cycles. Thirty-seven patients (48 per cent) conceived, six of them on two occasions, making 43 pregnancies of which 31.6 per cent were multiple. Five per cent of all treatment cycles were complicated by mild, and 0.62 per cent by severe hyperstimulation. The factors involved in achieving a satisfactory pregnancy rate whilst avoiding complications are discussed. Most complications occurred during the first cycle in which the rise in oestrogen excretion suggested follicular development and human chorionic gonadotrophin (HCG) was given (the "first effective" treatment cycle). In such cycles the risk of hyperstimulation rose sharply when the day 6 urinary total estrogen level was above 150 mug. per 24 hours and the multiple pregnancy rate was increased by a large dose of HCG.


Tubal pregnancy after sterilization.

Tubal pregnancy after sterilization. Twelve cases of tubal pregnancy after sterilization operations are reported and the pathogenesis is discussed. Diagnosis is sometimes delayed because of the history of previous sterilization. It is paradoxical that the diagnosis of an intrauterine pregnancy after tubal sterilization is rarely in doubt yet the possibility of the fertilized ovum implanting in a tube known to have been damaged is sometimes overlooked. It is suggested that bilateral salpingectomy is the correct operative treatment for this condition.


Successful pregnancy after conservative surgery and radiotherapy for disgerminoma of the ovary. A 19-year-old patient was treated for disgerminoma of the right ovary by right salpingo-oophorectomy and pelvic radiotherapy in 1967. The left ovary was shielded after the first two treatments and the patient has since conceived three times and has shown no evidence of recurrence of the tumour.


Engagement of the fetal head in primigravidae and its relationship to duration of gestation and time of onset of labour. In a retrospective survey on the time of engagement of the fetal head in 462 unselected consecutive primigravidae it was seen that by the end of the 37th week of gestation (259 days), engagement had occurred in only 23 per cent of them. The highest rate of engagement was from 39 to 40 weeks of gestation (273 to 280 days) and in 50 per cent of patients, engagement occurred between 38 and 42 weeks (266 to 294 days). The mean interval between engagement and delivery was 1.39 weeks (9.7 days), the median was 7 days, and the mode was less than 7 days. In 80 per cent of patients the engagement-delivery interval was less than 14 days. These findings strongly suggest that in the majority of primigravid patients the fetal head does not engage between 36 and 38 weeks of gestation (252 to 266 days) as is commonly believed.


Cytological diagnosis of a primary malignant melanoma of the vagina. The cytological appearances of a primary malignant melanoma of the vagina are described. A diagnosis of melanocarcinoma, made from smears showing malignant cells with melanin pigment in their cytoplasm, was confirmed histologically.


Concurrent ovarian and normal intrauterine pregnancy. A patient with concurrent ovarian and intrauterine pregnancy is described. Certain features were unique.


The origin of amnitoic fluid lecithin.

The origin of amnitoic fluid lecithin. Lecithin has been measured in amniotic fluid, pharyngeal aspirate, fetal and maternal plasma and fetal membranes from the same pregnancy. In the amniotic fluid from a term pregnancy 79 per cent of the lecithin is found in the reconstituted precipitate of centrifuged fluid. It is suggested that the lecithin of amniotic fluid may originate from sources other than fetal lung and that lecithin concentration is therefore a measure of overall fetal maturity.


Fetal heart rate monitoring during breech labour. Forty-two breech deliveries were monitored throughout labour. Serious asphyxia at birth occurred in 15 babies, in every case preceded by abnormalities in the fetal heart rate. Interpretation of the records implicated umbilical cord compression as the likely cause of asphyxia.


Biochemical and haematological changes during the induction of labour at term with oxytocin, prostaglandin E-2 and prostaglandin F-2alpha. A total of 75 patients had labour induced near term using intravenous oxytocin, prostaglandin E-2 or prostaglandin F-2alpha. Biochemical and haematological investigations were performed to assess hepatic, renal and adrenocortical function and changes in platelet adhesiveness. In doses which were similarly effective in inducing labour, both prostaglandins were without the water-retaining effect of oxytocin.  Adrenocortical stimulation was greatest with oxytocin. No difference between these agents was observed for the other measurements made.


Effect of frusemide on fetal urine production. Hourly fetal urinary production rate (HFUPR) was measured by an ultrasonic technique in 12 antenatal patients before and after intravenous administration of 60 mg. of frusemide. In 8 patients pregnancy was normal and in 4 there was clinical evidence of fetal growth retardation. The duration of pregnancy varied from 32 to 41 weeks. The increase in HFUPR was 80 to 150 per cent in normal patients and 95 to 131 per cent in patients with fetal growth retardation. The increase in HFUPR seemed to have no impact on fetal circulatory volume, as the fetal heart rate stayed normal throughout the study period.


Dilatation of the parous non-pregnant cervix. In vitro cervical dilatations were carried out on 33 uteri removed by hysterectomy for conditions not involving the cervix from parous women in their reproductive span of life. A specially designed force measuring dilator was used for all the tests. The analysis of the results involved the application of a thick-walled cylindrical stress determination technique. Using this approach it was possible to calculate quasi-elastic moduli for different degrees of dilatation and obtain theoretical distributions of radial and circumferential stress across the cervical wall. These results indicated a yielding of the cervical tissue at about 9 to 11 mm. dilatation in a significant number of cases.


The relation between maternal and cord serum iron levels and its effect on fetal growth in iron deficient mothers without malarial infection. In this relatively iton deficient population both multiparous and primiparous pregnant women had similar serum iron levels. The mothers' levels had a linear correlation with the cord serum levels, but the lowest mean cord levels were above 100 mug./dl. maternal and cord blood haemoglobin levels,birthweights and placental weights showed no constant relation to the serum iron levels, and  even in anaemic mothers the fetus was relatively non-asaemic with a haemoglobin level aroun 13 g./dl. In non-anaemic mothers  the cord blood haemogloin rose rapidly and progressively.


Uterine activity in  induced labour.

Uterine activity in  induced labour. A study was made of the characteristics of oxytocin-induced labour. Twelve patients with, and eight without membrane rupture were closely matched for factors likely to influence the character and duration of labour. In both groups uterine activity increased until a stable state was achieved loowing which there was little alteration until delviery. The duration of labour was shorten in the group with ruptured membranes as compared with intact membranes although, paradoxically, the uterineactivity tended to be greater when the membranes were intact.


The uterine and cardiovascular effects of salbutamol and practolol during labour. Intravenous salbutamol, a beta-adrenoceptor stimulant, given to nine patients in normal  labour, with continous monitoring of uterine activity and of the maternal and fetal cardiovascular systems, was shown to decrease uterine activity significantly; maternal and fetal heart rates were significantly increased, and maternal systolic  and diastolic arterial pressures were significantly decreased  during the infusion, although no treatment had to be discontinued because of these effects. Apart from worsening of low back pain during the infusion in one patients, subjective side-effects were trival. With the salbutamol infusion continued at an effective maintenance rate, the carioselective beta-adrenoceptor blocking drug, practolol, given intravenously, reduced the maternal heart rate (although not significantly) but it did not alter the fetal heart rate; it also appeared to interfere transiently with the inhibiting action of salbutamol on uterine activity, but cerevical dilatation was arrested until the salbutamol infusion was discontinued. At least in five patients, labour remained suppressed until oxytoxin was infused intravenously.


The effects on platelet aggregation of oral prostaglandin E2 used for the induction of labour. Secondary ADP induced platelet aggregation was inhibited in 17 patients after the administration of oral prostaglandin E2 tablets for the induction of labour. Primary ADP induced aggregation was shown to be inhibited by oral prostglandin E2 when the platelets responded to the lower concentration of 0-5 mug/ml of ADP. This inhibition was not detectable when the higher concentration of 1-0 mug/ml ADP  was  necessary. There would appear to be a decreased risk of thrombosis initiated by platelet aggregation as a result of using oral PGE2 tablets for inducing labour.


Changes in plasma non esterified fatty acids and serum glycerol in pregnancy. Changes in plasma nonesterified fatty acids (NEFA) and serum glycerol in pregnancy were examined in a semi-serial study of 85 women. A preliminary study showed that it was almost impossible to achieve standardized conditions for sampling, and the compromise of taking a single venous sample without stasis, after 30 minutes rest and about 12 hours fasting, was adopted. Although there were large individual variations the patterns of change were, in general, the same for NEFA and glycereo although the two were not closely correlated. There was no +convincing change before 30 weeks of pregnancy but both increased in the last ten weeks, fell sharply in the first week post partum, and then rose again to late pregnancy levels by 6 weeks post partum before falling to non-pregnant levels by between 3 and 6 months post partum. Those patterns of change are in broad accordin with changes of fat storage and lipolysis associated with the reporoductive cycle. Changes in the patterms of NEFA were triviax the only significant alteration was a small rise in the proportion of C16:0 (palmitic) in late pregnancy.


Carbohydrate-induced lipogenesis in the human placenta of normal and diabetic pregnancies. Using  in vitro incubation, human placental slices have been shown to synthesize lipid from 14-C fructose, but to a lesser extent than from 14-C glucose. Diabetic placentae did not incorporate more of either sugar into lipid than did placentae from normal pregnancies; nor did insulin in the incubation medium enhance lipogenesis. There was a correlation between the extent of incorporation of 14-C fructose and 14-C glucose into triglyceride, suggesting a linked enzyme system for phosphorylation of the two hexoses.


Rate of fall in plasma progesterone and time to abortion following intra-amniotic injection of prostaglandin F2alpha, with  or without urea, in the second trimester of human pregnancy. Progesterone concentrations have been determined in a total of 175 peripheral plasma samples obtained serially from 20 women undergoing mid-trimester abortion. In ten patients abortion was induced in intra-amniotic administration of 50 mg. of prostaglandin F2alpha (PGF 2alpha) alone; in ten others it was induced with 50 mg. of intra-amniotic PGF2alpha with 80 g. of urea. Mean instillation-abortion intervals were 29-0 hours in the group receiving PGF2alpha alone and 12-1 hours in the groups receiving PGF2alpha and urea. In eight of the patients treated with PGF2alpha and urea, and in four of those treated with PGF2alpha alone, there were significant decreases in progesterone level (determined by calculation of correlation coefficients) during the instillation-abortion iterval. When plotted against instillation-abortion times, calculated rates of decease in peripheral plasma progesterone levels showed a statistically significant regression (p smaller than 0-05). This indicates that progesterone levels drop most rapidly in patients with shorter instillation-abortion times. There was no relationship between initial progesterone levels and instillation-abortion times.


Effect of protein hormones and steroids on tissue cultures of endometrial carcinoma. Neoplastic tissue was obtained from 30 patients with endometrial carcinoma and the response of this tissue to various hormones was studied in vitro by histological methods and by measuring the incorporation of 3-H thymidine into DNA. Progesterone and synthetic progestogens had a consistently adverse effect on tissue survival in vitro while pregneolone  had a beneficial effect.


Myocardial infarction and rheumatic heart disease in pregnancy. Case report. Myocardial infarction is a rare complication of pregnancy, only 45 proven cases having been reported. In only one patient was rheumatic heart disease an associated factor (Goldberger and Pokress, 1950).


X-linked retinitis pigmentosa.

X-linked retinitis pigmentosa. Of 107 consecutive patients with genetically-determined retinitis pigmentosa, 23 were provisionally diagnosed as having inherited the disease in an X-linked fashion. 42 affected males and 61 females were examined, and from the data obtained the following conclusions were drawn: (1) X-linked retinitis pigmentosa exists and is distinct from choroideremia. (2) In contrast to the results of previous surveys, X-linked retinitis pigmentosa is a common form of this disease and over 20 per cent. of retinitis pigmentosa is probably transmitted in an X-linked manner. (3) (a) In contradistinction to the findings of previous investigators, most if not all adult heterozygous females have detectable degenerative changes in the ocular fundus. (b) The ocular changes in heterozygous females are most easily detected by fundus examination, visual field testing, dark adaptation measurements, and estimation of retinal rhodopsin concentration. The single most frequent abnormality is peripheral retinal pigment epithelial atrophy, which is found in all adult heterozygous females. (c) The pattern of retinal dysfunction in heterozygous females, and in particular preservation of the ocular electrical responses, suggests that the disease in women is qualitatively different from that in men and in other genetic forms of retinitis pigmentosa. There is some evidience that the disease in heterozygous women is patchy. (d) Degeneration in heterozygous females is usually symmetrical, but great variation was found in the severity of degeneration amongst heterozygotes of similar ages. No non-genetic influences were found to account for this. No evidence came to light by which the importance of X-chromosome inactivation could be assessed in determining the phenotype of heterozygous women. (4) No evidience is available to determine the number of X-linked genes transmitting the disease.


Plasma vitamin A and beta-carotene in retinitis pigmentosa. Plasma levels of vitamin A and beta-carotene were measured by spectrophotometry in 39 patients with genetically determined retinitis pigmentosa, No  difference was found between the levels in these patients and the levels in a control group.


Miotics in closed-angle glaucoma.

Miotics in closed-angle glaucoma. The use of intravenous Diamox with variable doses of Pilocarpine was investigated in the treatment of primary closed-angle glaucoma. It was concluded that one drop of Pilocarpine 3 to 4 hrs after intravenous Diamox is the only parasympathomimetic drug necessary to terminated an acute attack.


Fellow eye in angle-closure glaucoma.

Fellow eye in angle-closure glaucoma. 118 consecutive patients with angle-closure glaucoma (ACG) were investigated. The primarily affected eye presented with acute ACG in 71 patients, with intermittent or chronic ACG in 41, and with acute ACG secondary to intumescent cataract in six. ACG was demonstrated in the fellow eye in 85 of the 118 patients (72 per cent.), including 72, 71, and 83 per cent. respectively of the three groups distinguished above. In 27 fellow eyes, ACG was demonstrable only by provocative testing. Eleven patients failed to return for follow-up investigation. A routine prophylactic peripheral iridectomy in the fellow eye is justified in patients who are unlikely to attend for follow-up examinations,, but may be avoided in about 23 per cent. of patients who attend regularly.


Chronic open-angle glaucoma and ocular hypertension. An epidemiological study. An epidermiological study of patients with chronic simple glaucoma or ocular hypertension suggests that the diagnosis of glaucoma is associated with a positive family history, acute blood loss, and diabetes mellitus. There was no association with other vascular disease or with smoking. Ocular hypertension was related to smoking habits but not to family history. The relationship of these variables to ocular hypertension/glaucoma status is discussed.


Acquired cupping of the optic nerve head in normotensive eyes. Clinical measurements of a horizontal cup/disc ratio were obtained among a sample of 160 normal twin subjects by stereoscopic biomicroscopy. Measurements of horizontal cup/disc ratio were normally distributed and theere was no significant association with sex, laterally, race, or refractive error in the mid-range. However, there was a significant association between size of the cup/disc ratio and age as well as intraocular pressure. After taking into account the correlation between age an intraocular pressure in normal eyes, the association between cup/disc ratio and each of these variable remained statistically significant. It is not always recoginized that the size of the normal physiological cup increases with age, and it was not previously observed that "normal" cupping occurs with age apart from the effect of pressure. The average effect of age on cup/disc ratio, amounting to an icrease of 0-0026 per year for this sample, is small. But, allowing for individual variation, some persons could show a recognizable acquired increase in cup/disc ration with age, even in the absence of glaucoma. The possibility of "normal"cupping occurring in association with age, pressure, or high myopia, alone or in combination, merits cosideration in the clinical evaluation of glaucoma suspects and  serves to re-emphasize the importance of an adequate central visual field examination in the diagnosis of glaucoma. Studies are needed on the differentiatin between "normal" and "morbid" moulding of the optic nerve head.


Atrophy of myelinated nerve fibres in the retina in optic neuritis. Atrophy of the nerve fibres in the retina visible by direct ophthalmoscopy using red-free illumination, together with corresponding scotomata in the visual fields, signified retrograde degeneration of retinal nerve fibres from multiple sclerosis. Defects in medullated retinal nerve fibres were continous with arcuate nerve fibre bundle defects. These myelin defects exemplify secondary demyelination of optic axons.


Prevention of blindness from glaucoma using the  King's College Hospital computerized problem orientated medical record. (1) Chronic glaucoma accounts for a high proportion of blindness which is preventable and calls for energetic action because existing knowledge is not applied as it should be because of the indifference of the Department of Health and Social Security to the glaucoma problem. (2) The condition is frequently insidious and advanced before being identified, and requires life-long supervision. (3) When diagnosed, the management of glaucoma is frequently inadequate and intermittent for a variety of reasons. (4) AtKing's College Hospital, a Glaucoma Centre has been initiated to supervise accurately and regularly  a large number of glaucoma patients, assisted by numerical recording and computer analysis. While we are fortunate in having a computer in the hospital, it is important to emphasize that the system can be operated without this facility,  either by employing manual methods, or by batch processing. It would be both possible and desirable to organize recording a nd analysis on a regional basis in collaboration with hospitals wishing to participiate. (5) Attempts are being made to improve the early diagnosis ofglaucoma by better communication between the hospital ophthalmologists, and other members of the medical, optical, and ancillary professions by lectures, demonstrations, and publications. (6) Research is always hampered by the absence of factual knowledge. It is planned to use fully the opportunity for research into glaucoma made possible by this basic organization. At present however, we consider it more important to carry out investigations into the problems of organizing the investigation, treatment, and follow-up of glaucoma patients than t o embark on a few individual projects of research. We are serously hampered in our work by shortage of funds for staff and facilities, but we look forward confidently to the  time when, with the essential support of the Department of Health, these methods will give  us access to the facts of glaucoma, which besides enabling us to give a high standard of treatment to our patients, will give us an unprecedented opportunity of wide ranging research into all aspects of this important disorder.


Role of non-drainage of subretinal fluid in re-operation for retinal detachment. In a study of 194 re-operations for retinal detachment in which one-third of the cases were managed without drainage of subretinal fluid, it was found that a number of failures occurred after non-drainage. Analysis of these failures shows that more than half could have been avoided if the correct choice of cases suitable for management without drainage of subretinal fluid had been made. It is felt that with careful attention to the criteria which are used to decide on the choice of surgical technique, the results obtained by the non-drainage method should be as good in the more complicated case as they have already been shown to be in the primary case. Because the drainage of subretinal fluid carries with it a risk of surgical complications and these complications may contribute to the development of massive preretinal retraction, management of the re-operation should be carried out without drainage of subretinal fluid whenever possible.


Angioid streaks. I. Ophthalmoscopic variations and diagnostic problems. Fifty-six patients with angioid streaks were evaluated ophthalmologically. Most had repeated fundus photography and fluorescein angiography during a follow-up period of 6 months to 7 years. The ophthalmoscopic variations and diagnostic difficulties which occurred were noted. In most instances, the angioid streaks were not initially recognized and the patient was referred with another diagnosis. In several cases, the peripapillary, macular, and peripheral changes seen with angioid streaks were found to simulate other better known fundus conditions, resulting in the erroneous diagnosis and improper treatment. In some cases, the angioid streaks were so subtle that they were overlooked and in others they were observed, but initially interpreted as something else. Because of the medical significance of angioid streaks, ophthalmologists should be aware of their variable features. These are discussed, with emphasis upon those subtleties which differentiate angioid streaks from other conditions which they may simulate. On the basis of these observations, an ophthalmoscopic differential diagnosis of angioid streaks is proposed.


Angioid streaks and traumatic ruptures of Bruch's membrane. Minor blunt trauma may cause typical haemorrhages and probably enlargement of breaks or new breaks in patients affected with the Groenblad-Strandberg syndrome.


Aqueous humour lactic dehydrogenase isoenzymes in retinoblastoma. LDH activity was determined in aqueous humour samples from 11 eyes (of 10 children), four of which contained retinoblastoma. Simultaneous serum LDH levels were also determined in eight of the children. There was no correlation between serum and aqueous humour LDH activity. Total aqueous humour LDH activity ranged from 0 to 99 i.u/l. in the seven eyes with non-neoplastic conditions. It was 56, 124, 158, and 1832 i.u./l. respectively, in the four eyes with retinoblastoma. In all four eyes the ratio of isoenzymes LDH5:LDH1 was greater than 5. The total aqueous humour LDH levels in retinoblastoma was neither consistently elevated, nor related to the total serum LDH. There was a characteristic LDH isoenzyme fractionation pattern which, it is suggested, may be present before the total aqueous humour LDH becomes elevated.


HL-A 27 and acute anterior uveitis.

HL-A 27 and acute anterior uveitis. 51 (30 men and 21 women) of 90 consecutive patients with acute non-granulomatous anterior uveitis were HL-A 27 positive. This frequency of 55-7 per cent compares with 8-2 per cent in controls. Twenty-three patients (18 men and 5 women) had in addition evidence of systemic disease, including ankylosing spondylitis, sacroiliitis and Reiter's syndrome, sometimes associated with psoriasis. Twenty-eight of 63 patients without evidence of systemic disease were HL-A 27 positive, suggesting that the uveitis in many of these cases has a similar aetiology to those with rheumatic disease. The uveitis associated with HL-A 27 is typically unilateral, associated with mechanical ptosis, and a painful diffusely red, photophobic, and lacrimating eye, generally lasting 3 weeks or more. Protein extravasation into the aqueous is considerable, cells are usually present in the aqueous and anterior vitreous, and keratic precipitates are never mutton fat in appearance. Recurrent episodes are characteristic. The association with HL-A 27 suggests that many if not most cases of non-granulomatous anterior uveitis have a close aetiological relationship to ankylosing spondylitis and Reiter's syndrome and it is likely that infective agents, leading to an unusual immunologically mediated inflammatory response in predisposed individuals, are involved. Ten patients with granulomatous anterior uveitis were HL-A 27 negative.


Medulloepithelioma involving the iris.

Medulloepithelioma involving the iris. The case is described of a benign medulloepithelioma in a child which originally was apparently confined to the iris.


Lacrimal immunoglobulins and complement quantified by counter-immunoelectrophoresis. Immunoglobulin and complement concentrations of tears were measured using a sensitive electroimmunodiffusion assay. In the 13 subjects tested, the predominant immunoglobulin was secretory IgA (mean concentration 107 mug/mg protein; standard error of mean 15; n equal 17). The B1C component of complement was measurable in half of the subjects studied. The data illustrate that it is possible to quantify exactly certain humoral indices which may be important in the host defense of the eye.


Urea in the treatment of epibulbar malignancies. Eight patients with epibulbar malignancies of the eyes were sucessfully treated with urea. One of them presented a malignant melanoma, one a tumour of Kaposi's disease, and six had squamous cell carcinomas. At least four out of these eight patients would have required enucleation of the globe or exenteration of the orbit as an alternative to urea therapy.


Peritoneal autografts in conjunctival replacement. Twenty-eight rabbits underwent conjunctival replacement with peritoneal autografts. The grafted peritoneum acted as a scaffold for the remaining healthy conjunctiva which gradually replaced the grafted peritoneum. Peritoneum is not an effective conjunctival substitute as it does not survive in its own right. I should like to thank Professor Norman Ashton, FRS for his most helpful guidance and constructive criticism in this work. My thanks are also due to Miss E. Robins for her enthusiastic technical support; to Mr. R. Alexander and Mr R. Keeble for their technical and photographic assistance, and to Miss E. FitzGerald for her secretarial help.


Interaction of carbohydrate binding sites on concanavalin A-agarose with receptors on adipocytes studied by buoyant density method. The interaction of concanavalin A (Con A) with isolated adipocytes was studied using Con A-Sepharose beads in the affinity binding buoyant density method previously used to study insulin receptors. Free Con A-Sepharose beads could be separated from the bound beads (cell-bead complexes) by sedimentation of the high density beads and floatation of the low density complexes. Sedimented and total beads could be determined by counting the radioactivity associated with [-125I]Con A coupled in tracer amounts to the beads. Various lines of evidence demonstrated the high specificity of binding. Soluble Con A, but neither insulin nor any of the other proteins tested, inhibited and reversed the binding of Con A-Sepharose to the cells. Whereas treatment of Con A- (and insulin-) derivatized beads with anti-insulin antiserum, and cells with trypsin, readily inhibited binding of insulin-Sepharose to cells, neither treatment inhibited Con A-Sepharose binding. According to the relative extents of inhibition and reversal of binding exhibited by 15 different carbohydrates, the saccharide binding sites on Con A-Sepharose appeared virtually identical with the known sites on free Con A. Protein-containing components of cell ghosts that were solubilized with Triton X-100 appeared to correspond to the Con A-Sepharose receptor sites on the basis of their ability to bind to Con A-Sepharose columns, be eluted with methyl alpha-D-mannopyranoside (MeMan) and be precipitated by the free lectin and redissolved by MeMan. According to (a) Normarski interference contrast microscopic examination of the topographical distribution of Con A-Sepharose beads and cells surrounding and bound to each other, and (b) absence of any apparent morphological changes in the cells due to binding, it is suggested that extensive clustering ("cap" or "macropatch" formation) of Con A receptors did not occur on the adipocyte as a consequence of the interaction of the cells with the Con A-Sepharose beads.


Circular dichroism of oligosaccharides containing neuraminic acid. In order to test the usefulness of circular dichroism in stereochemical and structural studies of oligosaccharides of glycoproteins, we measured the circular dichroism (CD) for N-acetylneuraminic acid (NAcNA) and several derivates. By acidic mathanolysis, we have prepared the deacetylated methyl ester, methyl glycoside of NAcNA, as well as a saponified product. Circular dichroism of these compounds allows us to assign the transition due to the amide chromophore. There is a carboxyl n-pi transition at about 220 nm which has a negative CD band associated with it for the beta-methocyneuraminic acid, but changes sign for the methyl ester (methyl (methyl beta-D-neuraminid)ate). We isolated the trisaccharides N-acetylneuraminyl-(2 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-D-glucopyranose [(2leads to 3)NAcN-Lac] as well as (2 leads to 6)NAcN-Lac by paper chromatography and compared the CD for each. The two isomers show similar but distinguishable CD patterns, with a weak negative band due to the carboxyl group centered at 225 nm and a stronger positive band at 200 nm containing contributions from both the amide and carboxyl groups.


Effect of selected anions and solvents on the electron absorption, nuclear magnetic resonance, and infrared spectra of the N-retinylidene-n-butylammonium cation. The specific conteranion and the solvent have been shown to regulate the electronic excitation energy of the N-retinylidene-n-butylammonium cation. Halogenated hydrocarbon solvents which can hydrogen bond with the anion shift the lambda-max to longer wavelengths, whereas the solvent dipole, acting as a bulk effect, shifts the wavelength-max to shorter wavelength. Here solvents which can donate two hydrogens for hydrogen bonding, such as cis- and trans-1,2-dichloroethylene and cis- and trans-1,2-dichlorocyclohexane, are used as solvents for the Cl-, Br-, and I- salts. As expected the cis solvents allow longer wavelengths than do the trans solvents. Results of nuclear magnetic resonance spectroscopy are shown to be in agreement with electronic absorption spectroscopy. The C-11 proton and the C-13 and C-9 methyl protons show a considerable downfield shift in the salts with respect to the Schiff base. Furthermore the same protons show a continuing downfield shift as the anions are exchanged from Cl-, Br- to I-. This is an agreement with the interpretation of greater positive charge delocalization as the anions are changed in the above manner. The infrared absorptions of the C - N group in the Schiff base and the protonated form are shown to be almost similar. This is rationalized by showing that the force constant can remain constant as the highly related factors bond order, bond distance, and the effective electronegativity are changed in a self-compensating manner.


Electron microscopy and physical characterization of the carcinoembryonic antigen. Carcinoembryonic antigen (CEA), a glycoprotein material purified from human tumors, has been visualized by electron microscopy. At neutral pH, it consists largely of relatively homogenous, morphologically distinctive twisted rod or cruller shaped particles, with dimensions 9 x 40 nm. The particle length is considerably diminished at pH 40.0, which correlates with a known diminution of charge. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular weight of 180,000 in the peak region of the CEA band for both 10 and 15% acrylamide. When native CEA was treated with neuraminidase, reduced, and alkylated, a relatively compact random coil was produced, whereas reduction and alkylation without neuraminidase treatment produced a less configuration, as determined by sedimentation studies and by electron microscopy. Electrophoretic migration, however, was apparently unaffected by reduction and alkylation. Thus the characteristic CEA particle appears by several lines of evidence to be substantially folded into a recognizably tertiary structural arrangement.


Immunogenicity of liposomal model membranes sensitized with mono(p-azobenzenearsonic acid)tyrosylphosphatidylethanolamine derivatives. Antibody formation and delayed hypersensitivity reaction. We have previously reported that hapten specific antibodies are produced in guinea pigs immunized with certain N-substituted phosphatidylethanolamine derivatives (either free or incorporated into liposomal membranes) in complete Freund's adjuvant. In this paper, we describe the synthesis of mono(p-azobenzenearsonic acid)tyrosylphosphatidylethanolamine (ABA-Tyr-PE). Immunication with this compound (either free or present in liposomes) not only results in the formation of anti-azobenzenearsonyl antibodies, but also confers cellular immunity as manifested by delayed hypersensitivity reactions elicited by challenge with either azobenzenearsonyl-bovine serum albumin or sensitized liposomes. Thus, ABA-Tyr-PE immunized guinea pigs differ from those immunized with azobenzenearsonyl-bovine serum albumin which produce anti-bodies but do not reveal a delayed reaction. Moreover, the ABA-Tyr-PE immunized animals differ from those immunized with mono(p-azobenzenearsonic acid)tyrosine; this substance has been shown by other investigators to confer cellular immunity without antibody formation in guinea pigs. However, the deacylated homolog of ABA-Tyr-PE (i.e., mono(p-azobenzenearsonic acid)tyrosylglycerophosphorylethanolamine) has the same immunological properties as mono(p-azobenzenearsonic acid)tyrosine. These observations justify the further exploitation of liposomal model membranes as novel immunogens that are able to elicit both cell and humoral mediated immune responses.


Evaluation of quantitative affinity chromatography by comparison with kinetic and equilibrium dialysis methods for the analysis of nucleotide binding to staphylococcal nuclease. The elution of staphylococcal nuclease on thymidine 3'-(p-Sepharose-aminophenyl phosphate) 5'-phosphate (nucleotide ligand of nuclease covalently bound to Sepharose 4B) was studied in the presence of a variety of soluble nucleotide ligands. The elution volumes of nuclease vary proportionally with matrix-bound ligand concentration (at constant soluble ligand concentration), inversely with soluble ligand concentration (at constant matrix-bound ligand concentration), and inversely with dissociation constant of soluble ligand (at constant concentrations of soluble and matrix-bound ligand). The variation of elution volume was related to an expression which described the competition of soluble and matrix-bound ligand for nuclease binding. Using this expression, values for dissociation constants were derived for nucleotide ligands in both the soluble and bound form. The values for soluble ligand were found to correspond closely to those obtained by either equilibrium dialysis or kinetics of inhibition of nuclease activity. Furthermore, a close correspondence was found between the values of dissocation constants for matrix-bound and soluble thymidine 3'-(p-aminophenyl phosphate) 5'-phosphate, thus defining the interaction of nuclease with the matrix-bound ligand as a process quite similar to that occurring in solution.


A two-state conformational transition of the extracellular ribonuclease of Bacillus amyloliquefaciens (barnase) induced by sodium dodecyl sulfate. Barnase, the extracellular ribonuclease of Bacillus amyloliquefaciens, is shown to undergo a reversible two-state conformational transition at 0.65 mM sodium dodecyl sulfate (SDS) AAT 37 DEGREES. The prinicipal evidence is based on the equivalence of two independent values of the SDS-barnase binding ratio; about 14 mol of SDS/mol of barnase. Both were derived from fluorometric titration data, one being based on simple conservation of SDS and the other on the use of Wyman's theory of linked functions. No SDS is bound to barnase at SDS concentrations below the transition region.


Productive and unproductive lysozyme-chitosaccharide complexes. Kinetic investigations. Flow and relaxation methods were used to study the kinetics of oligosaccharides binding to lysozyme and the pre-steady-state kinetics of the lysozyme-catalyzed, hydrolysis of chitohexose. The minimal mechanism demonstrated, and the kinetics parameters pertaining to the elementary steps, allow interpretations of previous equilibrium and steady-state kinetic measurements which had yielded only complex constants, reflecting both productive and unproductive lysozyme-substrate complexes. In contrast to previous assumptions, the data presented in this paper provide evidence for "stable" productive lysozyme-substrate complexes. Our proposed mechanism utilizes structural information and accounts for the difference in efficiency of lysozyme-catalyzed hydrolysis of chitopentose and chitohexose.


Direct covalent mercuration of nucleotides and polynucleotides. Nucleotides of cytosine and uracil are readily mercurated by heating at 37-50 degrees in buffered aqueous solutions (pH 5.0-8.0) containing mercuric acetate. Proton magnetic resonance, elemental, electrophoretic, and chromatographic analyses have shown the products to be 5-mercuricytosine and 5-mercuriuracil derivatives, where the mercury atom is covalently bonded. Polynucleotides can be mercurated under similar conditions. Cytosine and uracil bases are modified in RNA while only cytosine residues in DNA are substituted. There is little, if any, reaction with adenine, thymine, or guanine bases. The rate of polymer mercuration is, unlike that of mononucleotides, markedly influenced by the ionic strength of the reaction mixture: the lower the ionic strength the faster the reaction rate. Pyrimidine residues in single- and double-stranded polymers react at essentially the same rate. Although most polynucleotides can be extensively mercurated at pH 7.0 in sodium or Trisacetate buffers, tRNA undergoes only limited substitution in Tris buffers. The mild reaction conditions give minimal single-strand breakage and, unlike direct iodination procedures, do not produce pyrimidine hydrates. Mercurated polynucleotides can be exploited in a variety of ways, particularly by crystallographic and electron microscopic techniques, as tools for studying polynucleotide structure.


Structure-function relationships and site of action of apamin, a neurotoxic polypeptide of bee venom with an action on the central nervous system. Specific chemical modifications of apamin have been used to study the residues involved in its toxic action. Transformation of Lys4 into homoarginine did not affect toxicity. Modification of the alpha-amino group of Cys1 and of the epsilon-amino group of Lys4 by acetic anhydride or fluorescamine decreased toxicity only by a factor of 2.5-2.8. Modification of the gamma-carboxylate of Glu7 with glycine ethyl ester in the presence of a soluble carbodiimide decreased toxicity by a factor of 2. Diethyl pyrocarbonate treated of the imidazole side chain of His18 decreased toxicity by a factor of 2.6. Thus none of these residues is essential for toxicity. However, combined modification of amino groups and of the imidazole side chain of His-18 completely abolished biological activity. Complete loss of toxicity also resulted from reduction and alkylation of both disulfide bridges, from chemical modification with cyclohexanedione of Arg-13 and Arg-14, and from removal of Arg-14 of acetylated apamin by digestion with trypsin. Incorporation of radioactive acetyl groups on both amino groups of apamin gave an active labeled toxin which has been used to localize the site of action of apamin in the spinal cord, principally in the lumbar part of the neuraxis.


A proton nuclear magnetic resonance study of the quaternary structure of human homoglobins in water. Proton nuclear magnetic resonance spectra of human hemoglobins in water reveal several exchangeable protons which are indicators of the quaternary structures of both the liganded and unliganded molecules. A comparison of the spectra of normal human adult hemoglobin with those of mutant hemoglobins Chesapeake (FG4alpha92 Arg yields Leu), Titusville (G1alpha94 Asp yields Asn), M Milwaukee (E11beta67 Val yields Glu), Malmo (FG4beta97 His yields Gln), Kempsey (G1beta99 Asp yields Asn), Yakima (G1beta99 Asp yields His), and New York (G15beta113 Val yields Glu), as well as with those of chemically modified hemoglobins Des-Arg(alpha141), Des-His(beta146), NES (on Cys-beta93)-Des-Arg(alpha141), and spin-labeled hemoglobin [Cys-beta93 reacted with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide], suggests that the proton in the important hydrogen bond between the tyrosine at C7alpha42 and the aspartic acid at G1beta99, which anchors the alpha1beta2 subunits of deoxyhemoglobin (a characteristic feature of the deoxy quaternary structure), is responsible for the resonance at -9.4 ppm from water at 27 degrees. Another exchangeable proton resonance which occurs at -6.4 ppm from H2O is a spectroscopic indicator of the deoxy structure. A resonance at -5.8 ppm from H2O, which is an indicator of the oxy conformation, is believed to originate from the hydrogen bond between the aspartic acid at G1alpha94 and the asparagine at G4beta102 in the alpha1beta2 subunit interface (a characteristic feature of the oxy quaternary structure). In the spectrum of methemoglobin at pH 6.2 both the -6.4- and the -5.8ppm resonances are present but not the -9.4-ppm resonance. Upon the addition of inositol hexaphosphate to methemoglobin at pH 6.2, the usual resonance at -9.4 ppm is shifted to -10 ppm and the resonance at 6.4 ppm is not observed. In the spectrum of methemoglobin at pH greater than or equal to 7.6 with or without inositol hexaphosphate, the resonance at -5.8 ppm is present, but not those at -10 and -6.4 ppm, suggesting that methemoglobin at high pH has an oxy-like structure. Two resonances (at -8.2 and -7.3 ppm) which remain invariant in the two quaternary structures could come from exchangeable protons in the alpha1beta1 subunit interface and/or other exchangeable protons in the hemoglobin molecule which undergo no conformational changes during the oxygenation process. These exchangeable proton resonances serve as excellent spectroscopic probes of the quaternary structures of the subunit interfaces in studies of the molecular mechanism of cooperative ligand binding to hemoglobin.


The conformation of glucagon: predictions and consequences. It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.


Salt dissociation of nuclear particles containing DNA-like RNA. Distribution of phosphorylated and nonphosphorylated species. Electrophoresis of proteins from nuclear particles containing DNA-like RNA gave a pattern with 45 bands. The possibility that some of these proteins arose by contamination with ribosomes, chromatin, or soluble nuclear proteins was examined and eliminated. The fate of the proteins of the particles was studied after partial dissociation with 0.25 and 0.70 M NaCl. The individual proteins were released progressively and in different quantities. A group of easily released species (75 and 95% removed with 0.25 and 0.70 M NaCl) was demonstrated. This group contained 8 species between 29,000 and 39,000 daltons which represented approximately one-half of the total number of molecules. It is suggested that they are bound to repetitive sequences of the RNA. At least 30 and 60% of the other proteins were released at 0.25 and 0.70 M NaCl, respectively. There were no specific proteins tightly bound to the RNA, unless the nature of the remaining species is different from that of the released ones of the same molecular weight. The phosphorylated proteins were more tightly bound to the RNA than the nonphosphorylated species of similar molecular weight. In several instances, the 32-P radioactivity was associated with quantitatively minor bands of proteins.


Metabolic fate of fatty acids in the carnitine cycle of brown adipose tissue mitochondria. Freshly isolated mitochondria from brown adipose tissue are uncoupled with respect to oxidative phosphorylation. When these mitochondria oxidize[U-minus 14-C] palmitic acid in the presence of malate the label is found in three major fractions. Polar lipids, rich in acyl carnitines, remain in the mitochondrial pellet. A large fraction, rich in tricarboxylic acid cycle intermediates, is exported to the suspending medium, as is a third, smaller fraction containing ketone bodies and beta-hydroxy-beta-methylglutaric acid. Prevention of oxygen uptake by addition of rotenone or antimycin prevents accumulation of cycle intermediates, increases formation of acyl carnitiness and increases beta-hydroxybutyrate relative to acetoacetate. Rotenone and antimycin do not prevent formation of labeled phosphatidylcholine. Partial suppression of oxygen uptake by benzene-1,2,3-tricarboxylic acid, amytal or malonate leads to results between these extremes. Addition of lysophosphatidylcholine had minimal effects on export of cycle intermediates, but increased formation of ketone bodies and particularly of acyl carnitines. The significance of lysophosphatidylcholine as an endogenous modifier of mitochondrial metabolism is discussed.


Further studies on the formation of cardiolipin and phosphatidylglycerol in rat liver mitochondria. Effect of divalent cations and the fatty acid composition of CDP-diglyceride. The divalent cation requirement for mitochondrial cardiolipin biosynthesis has been further investigated. The relative order of divalent cation activity was Co-2+ greater than Mn-2+ greater than Mg-2+. Cardiolipin was not formed in the incubations with Zn-2+, Fe-2+, Cu-2+, Hg-2+, and Ca-2+. Cardiolipin synthesis in the presence of optimal cincentration of Co-2+ was inhibited by Ca-2+. A series of CDP-diglycerides was synthesized having differences in fatty acid chain lenth and degree of unsaturation. These compounds were tested in mitochondrial cardiolipin and phosphatidylglycerol synthesis. Although there were some minor differences between phosphatidylglycerol and cardiolipin synthesis, in general, saturated shorter chain CDP-diglycerides (dilauroyl and dimyristoyl) were better substrates than the longer chain dipalmitoyl and distearoyl homologues. Introduction of double bonds into distearoyl CDP-diglyceride resulted in more rapid rates of synthesis (e.g. dioleoyl and dilinoleoyl CDP-diglyceride). Significance of the results is dicussed with regard to possible mechanisms of linoleic acid incorporation into rat liver cardiolipin.


The relative utilization of the acyl dihydroxyacetone phosphate and glycerol phosphate pathways for synthesis of glycerolipids in various tumors and normal tissues. Rates of phosphatidate synthesis from dihydroxyacetone phosphate via acyl dihydroxyacetone phosphate or glycerol phosphate are compared in homogenates of 13 tissues, most of which are deficient in glycerol phosphate dehydrogenase (EC 1.1.1.8). In all tissues examined, dihydroxyacetone phosphate entered phosphatidate more rapidly via acyl dihydroxyacetone phosphate than via glycerol phosphate. Tissues with a relatively low rate of phosphatidate synthesis via glycerol phosphate, showed no compensating increase in the rate of synthesis via acyl dihydroxyacetone phosphate. The rates at which tissue homogenates synthesize phosphatidate from dihydroxyacetone phosphate via glycerol phosphate increase as glycerol phosphate dehydrongenase increase. Both glycerol phosphate dehydrogenase and glycerol phosphate: acyl CoA acyltransferase (EC 2.3.1.15) are more active than dihydroxyacetone phosphate : acyl CoA acyltransferase (EC 2.3.1.42). Thus, all the tissue homogenates possessed an apparently greater capability to synthesize phosphatidate via glycerol phosphate than via acyl dihydroxyacetone phosphate, but did not express this potential. This result is discussed in relation to in vivo substrate limitations.


Acyltransferase activities in rat lung microsomes. Some properties of acyl-CoA:1-acyl-sn-glycero-3-phosphorylcholine acyl-transferase in rat lung microsomes wed moiety of acyl-CoAs, quite different values were obtained on the Michaelis constant, the maximal velocity, and the activation energy. Moreover, the incorporation of fatty acid from an acyl-CoA was affected in a different manner by the addition of other acyl-CoAs. These results suggested that there are at least two different acyltransferases which are tentatively termed as follows: (1) palmitoyl-CoA: 1-acylglycerophosphorylcholine acyltransferase; and (2) arachidonoyl-CoA: 1-acylglycerophosphorylcholine acyltransferase. A low Km value, a low maximal velocity, and a low value of the activation energy were obtained for the former activity. The activity is readily inhibited by the addition of other acyl-CoAs and also at the higher concentration of palmitoyl-CoA itself. While a high Km value, a high maximal velocity, and a high value of the activation energy were obtained for the latter activity. The activity is not affected by the addition of palmitoyl-CoA or oleoyl-CoA and only slightly inhibited by linoleoyl-CoA, which indicates a high substrate specificity for polyenoyl-CoA especially for arachidonoyl-CoA. It seems that the present result, together with the previous findings obtained in slice experiments and in in vivo studies, do not support the idea that palmitoyl-CoA : 1-acylglycerophosphorylcholine acyltransferase participates in the main pathway for the formation of dipalmitoyllecithin in lung.


Molecular weight of base plates of bacteriophage T4D. Highly purified base plates of bacteriophage T4D were obtained from lysate of gene 19 am mutant of this phage by differential centrifugation and sucrose gradient. Base plates were studied by means of high speed sedimentation equilibrium. The molecular weight determined by this method is (6.7 plus or minus 0.2)-10-6.


Structure-activity relationships of the vasopressor activity of canine peptide-A from fibrinogen. Canine peptide-A was demonstrated to be an active vasopressor substance. The effect of intravenous injection of canine peptide-A was a prolonged rhythmic vasoconstriction and is unlike any previously described vasopressor peptides. The intravenous administration of canine peptide-A resulted in a maximum increase in mean blood pressure of 19.0 mm of mercury at a dose level of 2.5-10-minus 2 mumol. This pressor effect exhibited a log proportionality to the injected dose of peptide. The duration of the vasopressor effect was 30 min or greater. A rhythmic variation in pressor response was also observed in all experiments. The canine AP and B peptides were inactive when tested under the same conditions. The relationship between structure and biological activity was investigated. Structural degradation of half of the peptide molecule was performed without loss of vasopressor activity. The canine peptide-A is postulated as assisting in local hemostasis by vasoconstriction.


Electron microscopy of low iodinated thyroglobulin molecules. Thyroglobulin molecules were studied in the electron microscope with negative staining technique. In a first series of experiments samples of thyroglobulin varying in iodine content from 0.5 to 0.03% were prepared from the thyroids of mice and rats kept on iodine-poor diets. All samples contained thyroglobulin molecules of the normal ovoid shape, not deviating in size or shape from molecules obtained from normal thyroids. However, in addition, another type of molecule having a cylindrical shape was observed in all samples. The proportion of these cylindrical molecules increased from a few per cent in the moderately iodine-poor thyroglobulin samples to more than 80% in the highly iodine-deficient thyroglobulin (0.03%). In a second series of experiments extremely iodine-poor thyroglobulin (smaller than 0.005%) was obtained from propylthiouracil-treated rats. In these preparations practically all molecules had a cylindrical shape. These samples also contained smaller particles interpreted to be dissociation products. The cylindrical molecules were of two types, one appearing compact and measuring 250 times 135 A (length times diameter) and the other appearing porous and having a length of 145 and a diameter of 205 A. It is concluded that the cylindrical molecules represent non- or low-iodinated thyroglobulin and it is suggested that the porous cylindrical molecule is an unfolded form of the compact cylinder.


The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction. Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.


Purification and properties of the anticoagulant principle of Trimeresurus gramineus venom. By means of DEAE-Sephadex A-50 column chromatography, Trimeresurus gramineus venom was separated into 12 fractions. Fraction 8 had marked anticoagulant action in the tests of whole blood clotting time, calcium clotting time and plasma prothrombin time. Fraction 8 was rechromatographed on Sephadex G-100, then on DEAE-Sephadex A-50 again, and finally on Sephadex G-100, and a single peak was obtained. The patterns of microzone and disc electrophoresis also showed a single band. A single symmetrical boundary with 1.70 Svedberg units was obtained by ultracentrifugation. The estimated molecular weight was 19 500. The isoelectric point was pH 4.5. Chemical analysis showed that the anticoagulant principle was a glycoprotein and that it was thermolabile. The anticoagulant activity of this purified principle was 3.5 times higher than that of the crude venom. Fraction 5 potentiated its anticoagulant activity to 10 times higher than that of the crude venom. This principle did not possess caseinolytic, tosyl-L-arginine methyl ester esterase, phospholipase A, phosphodiesterase, alkaline phosphomonoesterase, fibrinolytic, hemorrhagic or local irritating activities. The purified anticoagulant principle did not destroy fibrinogen, induce fibrinolysis, inactivate thrombin nor interfere with the interaction between thrombin and fibrinogen. However, a marked inhibition of prothrombin activation was caused by the anticoagulant principle. The inhibition of prothrombin activation was not due to the destruction of prothrombin or its activation factors, but due to an interference in the interaction between prothrombin and its activation factors because of the reversible binding of these factors with the anticoagulant principle of the venom.


The cold-insoluble globulin of human plasma: studies of its essential structural features. These investigations were directed at furnishing information on the essential structural features of the cold-insoluble globulin of human plasma. Amino acid and carbohydrate analyses showed that it is a glycoprotein (1.2% sialic acid, 1.8% hexose, 2.1% hexosamine) containing all of the amino acids usually found in proteins. Circular dichroic spectral analysis suggested that cold-insoluble globulin contained a very high proportion of beta-structure; no evidence for the presence of alpha-helix was found. Sedimentation velocity experiments at pH 7.0, in the presence or absence of dithiothreitol, plus related gel electrophoretic experiments at pH 8.4, indicated that the integrity of certain disulfide bridges was necessary for its solubility under "physiologic" conditions. In experiments in urea-containing solution, two sedimenting peaks were observed. The major one, amounting to more than 95% of the total, had an s20,w of 5.6 S, the minor peak had an s20,w of 7.3 S. Following disulfide bridge reduction a single symmetrical peak of 3.9 S was formed. Such behavior suggested that cold-insoluble globulin is a multichain molecule whose subunit chains are linked by disulfide bridging. Strong support for this conclusion was obtained from electrophoretic analyses in gels containing dodecylsulfate, in that cold-insoluble globulin manifested an increased rate of migration after reduction of disulfide bridges. The reduced cold-insoluble globulin band could be resolved into a closely spaced doublet, the components of which had molecular weights of 220 000 and 215 000, respectively. Since in sedimentation equilibrium experiments the molecular weight of the unreduced molecule was estimated to be 450 000, values in this range for the size of the subunit chain suggested that each cold-insoluble globulin molecule is composed of two covalently linked chains. The nature of the size heterogeneity of the reduced subunit chains is uncertain. However, the finding of a single type of NH2-terminal sequence ([Glu-Ala) in cold-insoluble globulin preparations, is consistent with the speculation that the smaller subunit may be a catabolic intermediate arising via release of peptide material containing the COOH-terminus of a parent chain.


Haemoglobin Arya: alpha 2-47 (CD5), aspartic acid yields asparagine. A new haemoglobin variant (haemoglobin Arya), is described from an Iranian female. The substitution is at residue 47 (CD5) of the alpha chain in which aspartic acid has been substituted by asparagine. The presence of haemoglobin Arya was not associated with clinical symptoms. This variant has normal stability at 50 degrees C, but is slightly unstable when tested at 55 degrees C.


Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Some suggested polymer subunit compositions. 1. Some of the individual members of the polymeric series of proteins from human haptoglobin types 2-1 and 2-2 were isolated by gel electrophoresis. By reacting this purified material with less than an equivalent amount of hemoglobin and analyzing the result by electrophoresis, the number of haptoglobin-hemoglobin complexes could be clearly counted. For the haptoglobin 2-1 series, the number of complexes formed was n+1, where n is the serial order, in decreasing electrophoretic mobility, of the haptoglobin polymeric form used. For the haptoglobin 2-2 series, the number of complexes was n+2. 2. For the first three members of haptoglobin 2-1 series, the haptoglobin-hemoglobin composition of the complexes was estimated from scans of the unstained gels. The data indicated that this series consists of 2,3,4... alpha beta haptoglobin subunits, each of which can combine with an alpha beta subunit of hemoglobin.


Hemoglobin Atlanta or alpha 2 beta 2 75 Leu-Pro (E19): an unstable variant found in several members of a Caucasian family. Hemoglobin Atlanta, alpha 2 beta 2 75 Leu-Pro (E19), has been found in several members of three generations of a Caucasian family living in metropolitan Atlanta. The abnormal hemoglobin is one of the nine unstable variants in which either a leucyl or an alanyl residue is replaced by a prolyl residue. These substitutions have been observed in the B, E, F, and G helixes of the beta chain and in the H helix of the alpha-chain. Hemoglobin Atlanta heterozygotes are mildly affected by the presence of this unstable hemoglobin.


Subunit structure of D-galactose dehydrogenase from Pseudomonas saccharophila. 1. D-Galactose dehydrogenase from Pseudomonas saccharophila (molecular weight 102 000) dissociates in 8 M urea into its subunits (molecular weight 25 000) which migrate in polyacrylamide gels, containing 8 M urea, as a single band. 2. The N-terminal residue determination by the dansyl method revealed only serine. 3. The C-terminal group determination with carboxypeptidase A and B indicated the sequence -Tyr-His-Leu. Leucine as the single C-terminal amino acid was confirmed by the tritiation method and by tritiation and subsequent degradation with carboxypeptidases. 4. The fragmentation of D-galactose dehydrogenase (24 mol methionine per mol enzyme) by CNBr resulted in six peptides, as detected in disc electrophoresis and substantiated by end group determination, indicating the identity of the subunits. 5. The treatment of D-galactose dehydrogenase (24 mol lysine and 52 mol arginine per mol enzyme) with trypsin and subsequent peptide mapping showed 21, perhaps 22 peptides, indicating a structure comprising four identical subunits.


On the stoichiometry between uncouplers of oxidative phosphorylation and respiratory chains. The catalytic action of SF 6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile). Titration of State 4 rat-liver mitochondria at pH 7.2 with the uncoupler 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) at various concentrations of mitochondria and using various substrates indicates that under optimal conditions less than 0.2 molecule of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile per respiratory chain is sufficient to induce complete uncoupling. This result suggests that there is not a stoichiometric relationship between uncoupler molecules and cytochrome c oxidase, involved in oxidative phosphorylation, or between the former and phosphorylation assemblies. Experiments on the release by 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile of azide-inhibited respiration of State 3 mitochondria and titrations with 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S13) of State 4 mitochondria at various mitochondrial concentrations confirm this conclusion.


Fluorescence lifetimes of alpha- and beta-carotenes. The fluorescence lifetimes of alpha- and beta-carotenes are both measured by a streak camera technique to be 55 plus or minus 10 ps at a concentration of 6 times 10- minus2 M in chloroform. Carotenes are present in chloroplasts at approximately this concentration.


Protein-protein interactions of light-harvesting pigment protein from spinach chloroplasts. I.Ca-2+ binding and its relation to protein association. The role of divalent cations in the regulation of the distribution of excitation energy between the two photosystems involved in green plants photosynthesis has led us to search for a better understanding of how such phenomena might occur at the molecular level. Since small changes in orientation of and distance between pigment molecules could greatly affect the distribution of excitation energy, we have decided to study the effects of ions on the light-harvesting pigment protein from spinach chloroplasts. The light-harvesting pigment protein is shown to have two types of binding sites for Ca-2+. Binding studies and analytical ultracentrifugation indicate that site I (K-d equals 2.5 mu-M, n equals 1.5-4.0 mu-mol Ca-2+ bound/mg chlorophyll) is lost as the protein associates. Site II (K-d equals 32 mu-M, n equals 9,5 mu-mol Ca-2+/mg chlorophyll) is not affected by the association of the protein. This site is responsible, however, for a further divalent cation-dependent association of the protein. The possible role of this protein in grana stacking and control of spillover is discussed.


Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the photosystem II. 1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments. 2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 A diameter. 3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.


On the steady-state electrical potential difference across the thylakoid membranes of chloroplasts in illuminated plant cells. The potential defference across the thyladoid membranes under steady-state saturating light conditions, measured with microcapillary glass electrodes, was found to be small as compared to the potential initially generated at the onset of illunimation. This result is discussed to be in agreement with quantitative estimates on the approximate magnitudes of the potential generating electron flux through the photo-synthetic electron transport chain and of the potential dissipating ion fluxes across the thylakoid membrane under steady-state conditions. It is concluded that a pH gradient of approx. 3-3.4 units is built up in the light across the membrane. The negative diffusion potential associated with this gradient is suggested to cause the transient negative potential observed in the dark after illumination.


The reduction of artificial electron acceptors at sub-zero temperatures by chloroplasts suspended in fluid media. 1. Chloroplasts can be suspended in aqueous/organic mixtures which are liquid at sub-zero temperatures with a good retention of the ability to reduce artificial electron acceptors. The reduction of ferricyanide and 2,6-dichlorophenolindophenol at temperatures above 0 degrees C is about 50% inhibited by 50% (v/v) ethylene glycol. Higher concentrations cause more extensive inhibition. 2. Different solvents were compared on the basis of their ability to cause a given depression of the freezing point of an aqueous solution. Ethylene glycol caused less inhibition of electron transport than glycerol, which in turn was found to be superior to methanol. 3. The reduction of oxidised 2,3,5,6-tetramethyl-p-phenylenediamine could be measured at -25 degrees C in 40% (v/v) ethylene glycol. Using an acceptor with a high extinction coefficient, methyl purple (a derivative of 2,6-dichlorophenolindophenol) it was possible to observe electron flow at temperatures as low as -40 degrees C in 50% (v/v) ethylene glycol. 4. From studies of the effects of the inhibitors 3(3,4-dichlorophenyl)-1,1-dimethylurea and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone it is suggested that electron flow from the donor side of Photosystem II to the acceptor side of Photosystem I can occur at temperatures at least as low as -25 degrees C. The ultimate electron donor is presumably water but it was not possible to demonstrate this directly.


Main structures of the Forssman glycolipid hapten and a Leb-like glycolipid of dog small intestine, as revealed by mass spectrometry. Difference in ceramide structure related to tissue localization. Two glycolipids of dog small intestine, one with Forssman activity and one with Leb-like activity, have been characterized by mass spectrometry of methylated, and methylated and reduced (LiAlH4) derivatives. The Forssman glycolipid was conclusively shown to be a pentaglycosylceramide with the carbohydrate sequence hexosamine-hexosamine-hexose-hexose-hexose-ceramide, and with sphingosine (dihydroxy base) as major long-chain base and normal fatty acids as the only fatty acids. The Leb-like glycolipid was a hexaglycosyl-ceramide with sequence fucose-hexose-[fucose-] hexosamine-hexose-hexose-ceramide and with phytosphingosine (trihydroxy base) as major long-chain base and only 2-hydroxy fatty acids as fatty acids. The difference of two hydroxy groups in the ceramide between the two glycolipids may be related to a different tissue localization. As shown by immunofluorescense study the Forssman activity was associated with the lamina propria and the Leb-like activity to the glandular epithelium of dog small intestine.


Drug-induced suppression of phospholipid synthesis in HeLa cells by inhibition of choline uptake. 2-(4-Anisidino)-4,6-bis (2-(diethylmethylammonium)ethylamino)-1,3,5-triazine diiodide was found to inhibit specifically the incorporation of [14 C]-choline into cold 5 percent trichloroacetic acid-insoluble materials in HeLa cells without affecting other vital areas of cellular metabolism. Further studies indicated that this drug did not affect any of the intracellular reactions leading to phosphatidylcholine formation; instead, the inhibition of lecithin synthesis was due primarily to the suppression of choline transport through the cytoplasmic membrane. The level of inhibition of the latter process was comparable to that observed for the inhibition of choline incorporation into cold 5 percent trichloroacetic acid-insoluble precipitates in whole cells.


Lipoprotein uptake by cultured human arterial smooth muscle cells. Human arterial smooth muscle cells growing in tissue culture, in contrast to rat cells, preferentially bind and take up large, lipid-rich lipoproteins (125I-labeled low density and very low density lipoproteins) in comparison to the known difference in the propensity of these two species to develop atherosclerosis.


Thermal transitions in the low-density lipoprotein and lipids of the egg yolk of hens. 1. Differential sanning calorimetry and light-scattering have been used to investigate temperature-dependent transitions in low-density lipoprotein and in lipids from hens' egg yolk. Yolks of different fatty acid composition were obtained by varying the dietary lipid and by adding methyl sterculate to the hen's diet. 2. Lipoprotein solutions in 50 percent glycerol/water gave characteristic melting curves between -25 degrees C and 50 degrees C, and on cooling showed increases in light-scattering between 10 degrees C and -20 degrees C. The temperatures at which major changes occurred depended on the proportions of saturated and unsaturated fatty acids. 3. The thermal transitions in the intact lipoprotein in glycerol solution were reversible, but with marked hysteresis. Lipid extracted from the lipoprotein did not show temperature  hystersis but the transition heats and melting curves similar to those of the intact lipoprotein. The results support the hypothesis of a "lipid-core" structure for low-density lipoproteins. 4. Scanning calorimetry of egg-yolk lecithins indicated a strong dependence of transition temperature on water content in the rane 3 percent-20 percent water. A rise in the mid-temperature of the liquid-crystalline to gel transition as the water content is lowered on freezing may be the primary event in the irreversible gelation of egg yolk and aggregation of lipoprotein.


A short-chain acyl-CoA: 1-alkyl-2-acyl-sn-glycerol acyltrasnferase from a microsomal fraction of the rabbit Harderian gland. The 3-position of the alkyldiacylglycerols from the pink portion of the rabbit harderian gland is occupied exclusively by isovaleric acid. We describe a microsomal 1-alkyl-2-acyl-sn-glycerol acyltransferase from this gland which specifically incorporates short-chain acyl-CoA's into the 3-position of alkylacyl-glycerols. The enzyme is most active in the presence of CoA esters with chain lengths similar to isovaleric acid and is inactive in the presence of acetyl CoA and long-chain acyl-CoA's. No evidence was found for an enzyme that would transfer long-chain acyl-CoA's to the same substrate. The specificity of this acyltransferase can account for the exclusion of long-chain acyl moieties from the 3-position of the alkyldiacylglycerols in the harderian gland of rabbits.


Interference with the transport of heparin-releasable lipoprotein lipase in the perfused rat heart by colchicine and vinblastine. The effect of pretreatment with colchicine or vinblastine on the lipoprotein lipase activity of rat heart was studied. Administration of colchicine or vinblastine 4 h prior to perfusion of the heart caused a very marked reduction in lipoprotein lipase activity released into the perfusate within 1 min of heparin perfusion. At the same time an increase in residual heart lipase occurred so that total lipoprotein lipase content of the heart (heparin releasable plus residual) did not change. The colchicine effect was dose and time dependent; no decrease in heparin-releasable enzyme activity occurred after only 30 min of pretreatment or upon addition of colchicine into the perfusate. These results indicate that colchicine did not impede enzyme synthesis or its release from the cell surface, but may have interfered with the transport of lipoprotein lipase from the site of its synthesis to the endothelial cell surface.